N371: Ca2+ imaging

What is calcium imaging?

An imaging technique that visualizes changes in intracellular Ca²⁺ concentration

What two types of resolution are required for Ca²⁺ imaging?

Temporal and spatial resolution

What are the three main ways to measure neural activity?

Electrical, optical, and molecular

What determines whether LTP or LTD occurs?

Magnitude and timing of Ca²⁺ signals

Why are molecular methods less useful for real-time activity?

They require post hoc processing

What happens when postsynaptic Ca²⁺ levels are low during plasticity?

Dephosphorylation of AMPARs → LTD

What happens when postsynaptic Ca²⁺ levels are high during plasticity?

CaMKII activation → AMPAR phosphorylation → LTP

What determines whether Ca²⁺ induces LTP or LTD?

Magnitude and timing of the Ca²⁺ signal

Why is Ca²⁺ signaling in the nucleus important?

It enables activity-dependent gene transcription

What happens when nuclear Ca²⁺ levels are low?

No BDNF transcription → LTP cannot be stabilized

What happens when nuclear Ca²⁺ levels are high?

BDNF transcription → long-term memory consolidation

What is BDNF broadly involved in?

Neuroplasticity, memory, and pain processing

What is a Ca²⁺-sensitive fluorophore?

A molecule whose optical signal changes when it binds Ca²⁺

What is a calcium chelator?

A molecule that binds Ca²⁺ tightly

How do Ca²⁺ dyes report intracellular calcium levels?

Ca²⁺ binding causes a conformational change that alters fluorescence

What kinds of fluorescence changes can Ca²⁺ dyes show?

Increased intensity or wavelength shift

What is aequorin?

A Ca²⁺-sensitive bioluminescent photoprotein. Ca²⁺ binding → conformational change → coelenterazine oxidation → blue photons

What is GFP and why does it glow without Ca²⁺?

GFP is a fluorescent protein that absorbs blue light and emits green light via photon absorption/emission, independent of Ca²⁺

What are genetically encoded calcium indicators (GECIs)?

Fluorescent proteins whose brightness changes with intracellular Ca²⁺, allowing activity measurement

Why do we report calcium signals as ΔF/F instead of raw fluorescence?

Baseline fluorescence varies across cells, so relative change is meaningful

What does ΔF/F represent biologically?

Change in Ca²⁺ relative to baseline → neural activity

What is GCaMP?

A genetically encoded calcium indicator that increases fluorescence when Ca²⁺ is present. Made of GFP + calmodulin (CaM) + M13 peptide

How does Ca²⁺ binding increase GCaMP fluorescence?

Ca²⁺ binds CaM → CaM binds M13 → conformational change → GFP deprotonates → fluorescence increases

Why is GCaMP especially good for population imaging?

Near-linear relationship between spike number and fluorescence

What is so grape about GCaMP? 😺

Can detect single APs + rapid upward spike shows neuronal firing!

Why are calcium dyes limited compared to GECIs?

Altough they are easier to introduce, dyes lack genetic targeting and are poor for long-term studies

What did GCaMP enable that dyes could not?

Imaging activity across species, cell types, and long timescales

Why does traditional fluorescence microscopy fail in deep tissue?

Light scattering, absorption, and out-of-focus fluorescence

Why is GFP excitation especially problematic in thick tissue?

Blue light scatters strongly and doesn’t penetrate deeply

Why are longer wavelengths better for deep tissue imaging?

Less scattering, less absorption, and less photodamage

Why dont short wavelengths work when trying to image deep tissue?

scatter a lot, don't reach deep neurons, and whatever fluorescence they excite doesn’t escape cleanly.

How does two-photon excitation work conceptually?

Two low-energy photons arrive simultaneously to excite one fluorophore

What problem does two-photon microscopy solve?

Imaging deep tissue with minimal damage and blur + only excites flourophores at the focal point!

What wavelength range is typically used for two-photon imaging?

Red near-infrared (NIR)

Why is two-photon excitation spatially precise?

High photon density exists only at the focal point

What is the “simultaneous rule” in two-photon microscopy?

Photons must arrive within femtoseconds to excite fluorescence

Why can’t we just use red-shifted fluorophores with one-photon imaging?

One-photon excitation still excites above and below the focal plane → blur

How does confocal microscopy reduce blur?

Uses a pinhole to block out-of-focus light

Why is confocal still limited for deep imaging?

Even with a pinhole, excitation still occurs outside the focal plane = blurred image + you cant be 100% sure where excitation came from

Why is it okay that two-photon microscopy doesnt use a pinhole?

excitation requires very high photon density (that only exists at the focal point) = signal location is unambiguous

Conceptually, what is the biggest advantage of two-photon microscopy over confocal?

Depth + specificity + reduced photodamage

Why are pulsed lasers required for two-photon microscopy?

Two-photon excitation needs very high photon density, which pulsed lasers provide

Why don’t pulsed lasers damage tissue despite high intensity?

Pulses are extremely brief, and gaps between pulses keep average power low

Why do high-NA objectives improve two-photon imaging?

They focus light into a tiny volume, increasing photon density at the focal point = Dramatically increases two-photon excitation

What are the three essential components of a two-photon microscope?

Ultrafast pulsed laser, scanning system, high-sensitivity detectors

Why is raster scanning needed in two-photon microscopy?

To reconstruct images line-by-line across the tissue

Why are photomultiplier tubes (PMTs) used?

To amplify extremely weak fluorescence signals

What does spine size correlate with?

AMPAR number and glutamate sensitivity

Why can’t astrocyte activity be measured with electrodes?

Astrocytes do not fire action potentials. Measured with Ca2+ signalling instead

What was the key breakthrough of the first in vivo Ca²⁺ imaging experiments?

They showed that neuronal activity could be visualized in a living brain

How did Ca²⁺ imaging expand our ability to study different cellular compartments?

It allowed activity measurement in axons, dendrites, and spines. It reveals how brain regions communicate with each other

Why is Ca²⁺ imaging especially powerful for studying dendrites and spines?

The same structures can be tracked functionally and structurally over weeks to months

What did spine-level Ca²⁺ imaging reveal in the visual system? (ORIENTATION lol)

Different spines on the same neuron respond differently to different orientations. This orientation preference revealed neuron’s overall prefernce is not the sum of individual spine preferences

Why can’t astrocyte activity be measured using electrodes?

Astrocytes do not fire action potentials. Measured using Ca2+ signals instead

How can Ca²⁺ imaging be combined with blood flow measurements? What happens to RBCs and glia/neurons during sensory stimulation?

By labeling red blood cells and imaging Ca²⁺ simultaneously. Neuronal/astrocytic Ca2+ increase and RBC speed increases = neurovascular coupling

What is mesoscale (large field-of-view) Ca²⁺ imaging?

Imaging activity from >10,000 neurons simultaneously! Allows us to measure many neurons in different regions

What problem do miniature microscopes solve in Ca²⁺ imaging?

Allows for imaging during freely moving, natural behavior + long term recording

Major limitation of miniature microscopes?

One-photon excitation only

What is fiber photometry?

Average Ca²⁺ activity from a population of cells (bulk Ca2+ imaging) through implanted (minimally invasive) optic fibers + genetically encoded sensors

What is the main tradeoff of fiber photometry?

Loss of single-neuron resolution (since its bulk imaging lol!)

Why was Ca²⁺ imaging essential for discovering how dendrites process sensory input?

It allowed simultaneous in vivo imaging of dendrites and soma. Electrodes cannot resolve many dendrites at once in behaving animals

What did Ca²⁺ imaging reveal about the soma in visual cortex neurons?

The soma performs spatial integration of dendritic inputs

What was discovered about sensory in neuron tuning over long timescales?

Individual neurons’ preferred orientation drifts over time. All neurons kinda drift at the same time so there is always enough cells to responds to a specific visual stimulus

How does stimulus exposure affect tuning drift?

More exposure → faster drift.

What was found when axons and dendrites shared sensory preferences?

Their synapses were larger = stronger synaptic communication = activity dependent synaptic strengthening

Why was it historically difficult to study activity in small brain nuclei? What was the solution?

Cant really see whats going on specifically using only electrodes. Genetically encoded Ca²⁺ sensors allowed recording from defined cell types!

What did Ca²⁺ imaging reveal about NE/ACh/5HT and arousal?

Norepinephrine and acetylcholine correlate with arousal, serotonin does not

What was discovered about dorsomedial hypothalamus (DMH) neurons?

Different axonal projections from the same region are active in different sleep states

Which DMH projections are active during non-REM sleep?

Axons projecting to anterior brain regions

Which DMH projections are active during REM sleep?

Axons projecting to posterior brain regions

What is the “labeled-line” model of behavior, why is it insufficient?

One cell group → one downstream target → one behavior. Not good, many brain regions participate in multiple behaviors

What did Ca²⁺ imaging reveal about epilepsy? Why is this important?

Seizures involve near-global neuronal synchronization. Seizures follow a predictable spatial seuqence = potential for targeted intervention

Why might Ca²⁺ imaging NOT be the right tool for some questions?

Poorer temporal resolution than electrophysiology

What is the primary quantity Ca²⁺ imaging estimates?

Action potential firing rate

Why is interpretation of Ca²⁺ signals sometimes ambiguous?

Ca²⁺ can arise from multiple intracellular sources, not just spikes

What key information does Ca²⁺ imaging NOT provide?

Membrane potential in the absence of spiking (subthreshold potential)

What is a major concern with introducing Ca²⁺ indicators into cells?

They may alter normal physiology